[go: up one dir, main page]

WO2003000907A2 - Transfection amelioree de cellules eucaryotes avec polynucleotides lineaires par electroporation - Google Patents

Transfection amelioree de cellules eucaryotes avec polynucleotides lineaires par electroporation Download PDF

Info

Publication number
WO2003000907A2
WO2003000907A2 PCT/EP2002/006897 EP0206897W WO03000907A2 WO 2003000907 A2 WO2003000907 A2 WO 2003000907A2 EP 0206897 W EP0206897 W EP 0206897W WO 03000907 A2 WO03000907 A2 WO 03000907A2
Authority
WO
WIPO (PCT)
Prior art keywords
cells
electroporation
mrna
transfected
egfp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2002/006897
Other languages
English (en)
Other versions
WO2003000907A3 (fr
Inventor
Gerold Schuler
Zwi N. Berneman
Viggo F.I. Van Tendeloo
Peter Ponsaerts
Isolde Strobel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nv Antwerpes Innovatiecentrum
Original Assignee
Nv Antwerpes Innovatiecentrum
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nv Antwerpes Innovatiecentrum filed Critical Nv Antwerpes Innovatiecentrum
Priority to JP2003507290A priority Critical patent/JP2004536598A/ja
Priority to CN028163621A priority patent/CN1701121B/zh
Priority to KR10-2003-7016741A priority patent/KR20040025690A/ko
Priority to EP02748810.5A priority patent/EP1397500B1/fr
Priority to BR0210936-0A priority patent/BR0210936A/pt
Priority to CA2451389A priority patent/CA2451389C/fr
Publication of WO2003000907A2 publication Critical patent/WO2003000907A2/fr
Publication of WO2003000907A3 publication Critical patent/WO2003000907A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4271—Melanoma antigens
    • A61K40/4272—Melan-A/MART
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention provides an improved method for gene delivery in eukaryotic cells by electroporation, preferably in human hematopoietic cells, particular dendritic cells.
  • the method of the invention is superior to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for gene delivery, including tumor antigen loading of dendritic cells.
  • DC Dendritic cells
  • DC can be transfected to comparable levels as compared to transduction by recombinant viruses, such as poxviruses Kim, C. J. et al., J. Immunother., 20:276-286 (1997)) or adenoviruses (Dietz, A. B., Vuk, P.S., Blood, 91:392-398 (1998)), while circumventing the drawbacks of viral vectors (Jenne, L et al., Gene Ther., 7:1575-1583 (2000); Jonuleit, H.
  • RNA has a short cellular half-life and lacks the potential to integrate into the host genome, thereby obviating safety concerns, e.g. insertional mutagenesis, in the context of clinical gene therapy trials (Lu, D. et al., Cancer Gene Ther., 1:245-252 (1994); Ying, H. et al., Nat. med., 5:823-827 (1999)).
  • this short cellular half- life may be disadvantageous since it may result in a relatively short protein expression.
  • Electroporation methods for the integration of cyclic polynucleic acids into "normal" cells are generally use the following reaction conditions (Van Tendeloo V.F.I et al., Gene Ther. 5:700-707 (1998); Van Tendeloo, V.F.I, et al., Gene Ther. 7:1431-1437 (2000); Van Bockstaele, D., Berneman, Z.N., Cytometry 41:31-35 (2000); Lurquin, P.F., Mol. Biotechnol. 7:5-35 (1997); Matthews, K.E. et al., Mol. Biotechnol., vol 48, Chapter 22, Ed.Nickoloff); Spencer, S.C., Biochem. and Biotechnol. 42:75-82 (1993)):
  • US patent no. 5,766,902 discloses an electroporation method for nucleic acid molecules, wherein the nucleic acid molecules are applied in or together with a ligand which binds to the target cell.
  • Said complex may comprise an endosomal disruption agent.
  • US patent No. 5,554,528 decribes the use of plasmids (i.e., cyclic DNA constructs) containing a toxin gene under the control of HIV elements for stable transformation of cell lines in order to block HIV replication when cells are infected.
  • Said patent mentions DNA transfection by electroporation (column 15, example 2) using electrical settings (250 ⁇ F; 220 to 290 V; 100 ⁇ l volumes, BioRad cuvettes and Gene Pulser®) which are not typical for plasmid electroporation, it does, however, not mention RNA transfection, let alone RNA electropration.
  • RNA transfection let alone RNA electropration.
  • only "normal" mammalian cell lines are electroporated, primary cells are not contemplated.
  • the present invention describes a method for high- efficiency non-viral transfection of Mo-DC as well as other types of dendritic cells (including CD34 + derived Langerhans cells and interstitial type DC) by mRNA electroporation correlated with effective loading of tumor antigens into different types of human DC.
  • the efficiency of the method of the present invention was compared with other transfection methods, such as lipofection and passive pulsing of mRNA as well as cDNA electroporation, and found to be highly superior. Furthermore, the effect of DC maturation on loading efficiency was investigated. An electroporation-based mRNA transfection protocol was developed which is suitable for highly efficient antigen loading in Mo-DC, as well as in 34-DC and 34-LC. This technique proved to be superior to mRNA lipofection or passive mRNA pulsing in terms of loading efficiency and subsequent activation of an antigen-specific CD8 + CTL clone.
  • the transfection efficiency in Mo-DC, 34-DC and 34-LC was at least 25, 6 and 3 times, respectively, more efficient as compared to plasmid DNA electroporation described in van Tendeloo, V.F.I, et al., Gene Ther., 5:700-707 (1998), and also superior to previously described mRNA electroporation. Also, such mRNA electroporation was superior to mRNA lipofection and passive pulsing. This increased transfection efficiency was translated in a superior biological effect in vitro, as confirmed by our CTL activation experiments, and could be used as a tool to investigate as to whether it results in a higher immunopotency in vivo (Porgador, A.
  • (2) a method for transfection of eukaryotic cells with one or more or a mixture of linear polynucleotides, preferably a method as defined in (1), which method comprises electroporation of a suspension containing the eukaryotic cells and the linear polynucleotides to be transfected with a soft pulse at 300 to 600 V for 100 ⁇ s to 1 ms.
  • composition or vaccine comprising transfected eukaryotic cells obtainable by the method as defined in (1) or (2) above;
  • transfected eukaryotic cells obtainable by the method as defined in (1) or (2) above for preparing an agent for immunotherapy, including induction of immunity or tolerance, tumour therapy, stem cell therapy, regenerative medicine, or tissue engineering;
  • transfected eukaryotic cells obtainable by the method as defined in (1) or (2) above as expression system for gene products encoded by the linear polynucleotides, or as detection system;
  • a method for immunotherapy or tumour therapy which comprises administering transfected eukaryotic cells obtainable by the method as defined in (1) or (2) above to the patient.
  • inventions (1) and (2) are applicable for loading human dendritic cells (DC) with antigens such as tumor antigens, which is a challenging approach for DC-based tumor vaccines.
  • DC dendritic cells
  • antigens such as tumor antigens
  • the present invention describes a cytoplasmic expression system based on mRNA electroporation to efficiently introduce genetic information into DC.
  • mRNA-electroporated DC retained their phenotype and maturational potential.
  • DC electroporated with mRNA encoding Melan-A strongly activated a Melan-A-specific cytotoxic T lymphocyte (CTL) clone in an HLA-restricted manner and were superior to mRNA-lipofected or -pulsed DC.
  • CTL cytotoxic T lymphocyte
  • Optimal stimulation of the CTL occurred when Mo-DC underwent maturation following mRNA transfection. Strikingly, a nonspecific stimulation of CTL was observed when DC were transfected with plasmid DNA.
  • Our data clearly demonstrate that Mo-DC electroporated with mRNA efficiently present functional antigenic peptides to cytotoxic T cells.
  • Figure 1 shows the flow cytometric analysis of transgene expression in K562 cells following EGFP mRNA electroporation.
  • AL K562 cells were electroporated with EGFP mRNA at 300 V, 150 ⁇ F or with EGFP plasmid DNA at 260V, 1050 ⁇ F (dashed line) as described in the Examples. Twenty-four hours post-electroporation, flow cytometric (FCM) EGFP analysis was performed to estimate transfection efficiency of mRNA electroporation (bold line) and plasmid DNA electroporation (dashed line). An overlay histogram representative of five independent experiments is shown. Non-electroporated cells (thin line) were used to determine background fluorescence. The Ml region indicates the EGFP-positive cell fraction. The percentage of EGFP + cells was 85% (bold line) and 50% (dashed line) following mRNA or plasmid DNA electroporation, respectively.
  • FCM flow cytometric
  • Figure 2 shows the FCM analysis of transgene expression following EGFP mRNA transfection in different types of DC.
  • Immature Mo-DC were cultured with GM-CSF and IL-4 and transfected at day
  • 34-DC (bottom) and 34-LC (top) were cultured as described in Materials and
  • Figure 3 shows the phenotypical analysis and maturation potential of mRNA- electroporated DC.
  • iMo-CD Ai Immature Mo-DC (iMo-CD) were transfected by electroporation with mRNA encoding EGFP and stained with phycoerythrin (PE)-labeled antibodies specific for CDla, HLA-DR and CD86 one day after electroporation (bottom). Untransfected iMo-DC (top) served as controls and isotype-matched antibodies were used to set quadrants. Results are representative of 3 experiments.
  • iMo-DC were transfected by electroporation with mRNA encoding Melan-A and directly stained with a PE-labeled CD80 antibody (bottom) or indirectly stained with a CD83 antibody (top).
  • a representative overlay histogram is shown in which the dashed line represents the control non-electroporated iMo-DC, the thin line the electroporated iMo-DC and the bold line represents electroporated iMo-DC that were allowed to mature for an additional 24 h following mRNA electroporation in the presence of TNF- ⁇ and LPS.
  • 34-DC 12 day-cultured 34-DC were transfected by electroporation with mRNA encoding EGFP and stained with PE-labeled antibodies specific for CDla, HLA-DR, CD86 and CD80 one day after electroporation (bottom). Untransfected 34-DC (top) served as controls and isotype-matched antibodies were used to set quadrants. Results are representative of 3 experiments.
  • FIG. 4 shows the mRNA-based antigen loading of Mo-DC.
  • the SK23-MEL melanoma cell line, HLA-A2 + Mo-DC pulsed with a Melan-A or irrelevant influenza peptide and HLA-A2-negative Mo-DC electroporated with Melan-A mRNA served as controls.
  • Figure 6 shows the outcome of plasmid cDNA-based antigen loading of 34-LC.
  • Figure 7 shows the result of electroporation of immature monocyte-derived cells, in particular, the phenotype of dendritic cells 48 h after electroporation with GFP- RNA.
  • the numbers in the lower right part of the quadrant indicate the EGFP- positive DC, the numbers in the upper right part show the EGFP+/CD83+ and EGFP+/CD25+ DC, respectively.
  • Figure 8 shows the transfection efficiency of and kinetics of EGFP expression i in dendritic cells following GFP-RNA-transfection using electroporation.
  • Figure 9 shows the results of EGFP RNA-transfection of monocyte-derived dendritic cells by electroporation.
  • A Contour plots showing the influence of voltage on cell size and granularity.
  • Figure 10 EGFP RNA-transfection of mature monocyte-derived dendritic cells by electroporation.
  • a and H show the transfection efficiency and kinetics of EGFP expression following GFP-RNA transfection of mature dendritic cells using electroporation.
  • B to G confirm that the phenotype of dendritic cells is maintained after electroporation with GFP-RNA.
  • Figure 11 FCM analysis of transgene expression in immature and mature DC after EGFP mRNA electroporation in non-frozen controls and after thawing of cryopreserved samples.
  • the dot plots show EGFP fluorescence on the x-axis and ethidium bromide staining on the y-axis. Analysis was performed on cells exhibiting a large forward scatter and large side scatter profile, in order to allow exclusion of contaminating autologous lymphocytes. Percentage of dead cells (upper left corner), viable EGFP+ cells (lower right corner) and viable EGFP- cells (lower left corner) is indicated based on the number of dots in the quadrant analysis.
  • A Dot plots show analysis of non-frozen iMo-DC 24 hours after mRNA electroporation (left), and of mRNA-electroporated iMo-DC 6 hours after thawing (middle) and 24 hours (right) after thawing.
  • B Dot plots show analysis of non- frozen mMo-DC 24 hours after mRNA electroporation (left), and of mRNA- electroporated mMo-DC 6 hours after thawing (middle) and 24 hours (right) after thawing.
  • EP electroporation.
  • Figure 12 Representative example of phenotypical analysis of non-frozen and frozen mRNA-electroporated immature and mature DC. Dot plots show FCM analysis of PE-labeled monoclonal antibodies directed against typical DC-markers including CDla, HLA-DR, CD80 and CD86 (y-axis). As controls to set quadrants, isotype-matched antibodies and a PE-labeled monoclonal CD14 antibody was used. Analysis of DC markers was done on viable EGFP- cells in control samples and on viable EGFP+ cells in mRNA-electroporated DC as shown by the EGFP fluorescence on the x-axis.
  • Thawed DC have the same upregulation of HLA-DR and CD80, but have lower levels of CD86 (Figure 12, C). This is probably caused by the fact that the frozen immature DC culture is dying 24 hours after thawing. Immature Mo-DC responded well to the maturation cocktail as seen by the upregulation of HLA-DR, CD80 and CD86 in mMo-DC as compared with the expression levels in iMo-DC ( Figure 12, A & D). However, the combination of mRNA electroporation and a maturation stimulus seems to be very potent in maturing DC, as this combination results in high level of HLA-DR, CD80 and CD86 expression ( Figure 12, E). Frozen mature DC that were electroporated show high level maturation marker expression after thawing ( Figure 12, F).
  • FIG. 13 Stimulatory capacity of cryopreserved mRNA-electroporated mature DC.
  • Cryopreserved matrix protein Ml mRNA-electroporated mature DC were used as stimulators for PBMC during a 6 day coculture.
  • Primed PBMC were then stimulated with T2 cells, pulsed with an MHC class I-restricted Ml immunodominant epitope, during a 6 hour coculture.
  • Antigen specific T cells in the primed PBMC culture were detected as shown by positive IFN- ⁇ production.
  • unpulsed T2 cells were used as stimulators and fresh PBMC as responders. Results are shown as mean + standard error.
  • the activated T cells in the primed PBMC culture Upon restimulation with peptide-pulsed T2 cells, the activated T cells in the primed PBMC culture produced IFN- ⁇ against the immunodominant matrix protein peptide. The specificity of this activation is shown by only background IFN- ⁇ production of the primed PBMC culture against unpulsed T2 cells. To show that these cultured PBMC were stimulated during the 6 day culture, the same experiment was done with fresh PBMC. After coculture with either T2 cells or T2 cells pulsed with the peptide, no IFN- ⁇ production was detected above background level (Figure 13).
  • Figure 14 Representative flow cytometric analysis of scatter profile and viability of short-term serum-free-cultured immature DC and poly-I:C-maturated DC of Example 6.
  • iMo-DC immature monocyte-derived DC
  • mMo- DC poly-I:C-maturated monocyte-derived DC
  • Upper dot plots show forward and side scatter profiles of all cells.
  • the Rl gate shows the percentage of DC in the cultures.
  • Lower dot plots show mortality by ethidium bromide staining within the cultured DC. (upper numbers, ethidium bromide-positive dead DC ; lower numbers, ethidium bromide-negative living DC).
  • the lower dot plots were gated on Rl (upper panel).
  • the data shown are from PBMC donor A.
  • the results are representative for PBMC from donors A, B, F for immature DC and A, B, C, D, E, F for mature DC,
  • Figure 15 Representative phenotypical analysis of short-term serum-free- cultured immature DC and serum-free-cultured poly-I:C-maturated DC of Example 6.
  • Figure 16 Allogeneic stimulatory capacity of short-term serum-free-cultured immature DC versus serum-free-cultured poly-I:C-maturated DC of Example 6.
  • Immature and mature short-term cultured DC (respectively iMo-DC and mMo- DC) were used as stimulators for allogeneic PBMC during a 7-day coculture.
  • primed PBMC were restimulated with PBMC from the DC donor during a 6-hour coculture.
  • Activated T-cells in the primed PBMC culture were detected as shown by IFN- ⁇ production against the target PBMC.
  • Results are shown as mean ⁇ standard deviation of two individual experiments for cultures initiated with immature DC (iMo-DC) and mature DC (mMo-DC). The significant difference is indicated with an asterisk. Results were obtained with PBMC from donors B and C.
  • FIG 17 Stimulatory capacity of short-term serum-free-cultured immature DC versus serum-free-cultured poly-I:C-maturated DC (Example 6).
  • Influenza matrix protein Ml peptide-pulsed immature and mature DC (respectively iMo-DC and mMo-DC) were used as stimulators for autologous PBMC during a 7-day coculture.
  • primed PBMC were restimulated with T2 cells, pulsed with a MHC class I-restricted influenza matrix protein Ml peptide (T2/M1), during a 6-hour coculture.
  • T2/M1 MHC class I-restricted influenza matrix protein Ml peptide
  • Figure 18 Stimulatory capacity of serum-free-cultured immature DC versus serum-free cultured poly-I:C-maturated DC.
  • Direct staining of IFN- ⁇ -secreting CD8+ T cells after restimulation with an influenza target (Example 6).
  • Influenza matrix protein Ml peptide-pulsed immature and mature DC (respectively iMo-DC and mMo-DC) were used as stimulators for PBMC during a 7-day coculture.
  • Primed PBMC were then restimulated for three hours with T2 cells pulsed with a MHC class I-restricted influenza Ml peptide or with an HPV E7 control peptide.
  • Dot plots show IFN- ⁇ -secreting cells within the CD8+ and CD8- lymphocyte population.
  • the numbers of IFN- ⁇ -secreting cells indicated on the dot plots are percentages of total lymphocytes. Results were obtained with PBMC from donor B.
  • Figure 19 Representative flow cytometric analysis of scatter profile, viability and EGFP expression of EGFP mRNA-electroporated monocytes short-term cultured to mature DC (Example 6). Monocytes, electroporated (EP, lower dot plots), or not (EP, upper dot plots), with EGFP mRNA were cultured for 2 days in AIM-V medium + GM-CSF. Maturation was induced by poly-I:C after 24 hours of culture.
  • A Scatter profile of the cultured mature DC.
  • B Ethidium bromide staining of the cultured mature DC. The dot plots were gated on Rl (scatter profile). The indicated numbers show the percentage of ethidium bromide-negative living DC.
  • Figure 20 Representative phenotypical analysis of monocytes electroporated with mRNA and short-term serum-free cultured to mature DC (Example 6). Flow cytometric analysis of PE-labeled monoclonal antibodies directed against DC and monocyte markers: CD14, CD80, CD86, HLA-DR and CD83. Monocytes, electroporated (EP, lower dot plots), or not (EP, upper dot plots), with EGFP mRNA were cultured for 2 days in AIM-V medium + GM-CSF. Maturation was induced by poly- .C after 24 hours of culture. Histograms show the level of marker expression (black overlay) against isotype control staining (dotted line). The data shown are from PBMC donor D. The results are representative for PBMC from donors C, D, E.
  • Figure 21 Stimulatory capacity of mRNA-electroporated monocytes short-term serum-free cultured to mature DC (Example 6).
  • Monocytes electroporated with influenza matrix protein mRNA, were cultured for 2 days in AIM-V medium + GM-CSF. Maturation was induced by poly-I:C after 24 hours of culture. These mature antigen-loaded DC were used as stimulators for autologous PBMC during a 7-day coculture. Afterwards, primed PBMC were restimulated during a 6-hour coculture with T2 cells, pulsed with a MHC class I- restricted influenza matrix protein Ml peptide (T2/M1). Antigen-specific T-cells in the primed PBMC culture were detected as shown by increased IFN- ⁇ production. As a control, irrelevant HPV E7 peptide-pulsed T2 cells (T2/E7) were used as stimulators.
  • T2/E7 irrelevant HPV E7 peptide-pulsed T2 cells
  • Results are shown as mean ⁇ standard deviation of three individual experiments for PBMC from donor B (indicated as A) and PBMC from donor C (indicated as B). Significant differences are indicated with an asterisk.
  • Figure 22 The results of the EGFP analysis of the transfected cells of Example 7 at 24 and 96 h is shown in Fig. 22A and B, respectively.
  • the phenotypic analysis of the transfected cells of Example 7 after 96 h (CD34/gated on CD45+ cells; CD 19/gated on DR+ cells; CD 14/gated on CD33+ cells; CD 4/gated on CD7+ cells) is shown in Figs 22C to F, respectively.
  • Figure 23 shows the results of the EGFP analysis and phenotypic analysis of the transfected embryonic stem cells of Example 8, without feeder (A) and with feeder (B).
  • Figure 24 shows the results of the EGFP analysis and phenotypic analysis of the PBMC electroporated according to the method of Example 9
  • Figure 25 mRNA-electroporation of Mo-DC at the ⁇ s-range (Example 10B)
  • FIG. 26 Influence of pulse form on transfection efficiency (Example 10C) Immature Mo-DC were electroporated for 500 ⁇ s at 400 V using the machines MULTIPORATOR ® (Eppendorf, Hamburg, Germany) and ECM830 ® (Genetronics BTX, San Diego, CA, USA) delivering exponential decay or rectangular pulses, respectively. Immediately after that terminal maturation was induced by addition of IL-16, IL-6, TNF- ⁇ and PGE 2 . Mature DC were electroporated at the same settings. Transfection efficiacy was determined by FCM analysis 2d post- electroporation. The dashed line shows the fluorescence of DC transfected with EGFP mRNA. The dotted line represents fluorescence of DC transfected with FluMl mRNA. The numbers in the figure indicate the mean fluorescence intensity (MFI).
  • MFI mean fluorescence intensity
  • FIG 27 Phenotypical analysis of Mo-DC 2d after mRNA-electroporation at the ⁇ s-range (Example 10C). Immature and mature Mo-DC were electroporated as described in Figure 26/Example IOC. The dashed line shows the red-fluorescence of Mo-DC stained with the monoclonal antibodies specific for CD83 and CD25, respectively. The dotted line represents the isotype control. The number in the figure indicates the mean fluorescence intensity (MFI).
  • MFI mean fluorescence intensity
  • Figure 28 Scale-up of the cell number per electroporation cuvette (Example
  • A The dashed line shows the fluorescence of Mo-DC transfected with EGFP mRNA.
  • the dotted line represents negative control.
  • an conventional electroporation apparatus which provides for an exponential decay pulse. It is moreover preferred that the electroporation is performed at a voltage from 100 to 500 V, more preferably from 200 to 350 V, most preferably from 250 to 300 V. It is also preferred that the capacitance is 100 to below 300 ⁇ F, preferably 150 to 250 ⁇ F.
  • the pulsing time is strongly dependent from the type of the tray (cuvette) and the amount of reaction mixture (cell suspension) in the cuvette and is generally below 50 ms, preferably below 40 ms.
  • the pulsing time is from 5 to 40 ms, preferably 1 to 25 ms, and most preferably 7 to 10 ms.
  • different voltage and pulsing times can easily be determined by the skilled artisan.
  • soft pulse electroporation devices are utilized. With such devices the following settings a voltage of 300 to 600 V and a time of 100 ⁇ s to 1 ms are utilized which are believed to correspond to a capacitance of below 300 ⁇ F (although, due to the use of eukaryotic cell suspensions; a correct conversion is not possible).
  • the pulse form provided by commercially available soft pulse electroporation devices may be a square wave pulse or an exponential decay pulse.
  • Preferred settings for the soft pulse devices are 350 to 450 V for 300 to 600 ⁇ s.
  • the concentration of the cells in the suspension is lxlO 3 to lxlO 9 cells per ml, preferably lxlO 5 to lxlO 9 cells per ml. Even more preferred are lxlO 5 to 5xl0 7 cells per ml, most preferably 1 to 4xl0 7 cells per ml.
  • linear polynucleotides to be transfected are preferably so-called "naked" polynucleodides, i.e. polynucleotides which are not complexed or stabilized by a ligand or the like.
  • Linear polynucleotides to be utilized in the present invention include, but are not limited to, modified or unmodified, defined or undefined DNA, RNA or DNA-RNA hybrids and all kinds of modified variants thereof.
  • the most preferred linear polynucleotides are mRNA.
  • the above DNA-RNA hybrides are particularly suitable to repair or modify genes (Stepehnson, J., JAMA 281 (2), 119-122 (1999)).
  • the concentration of the polynucleotides to be transfected is lxlO "7 to lxlO "5 mmol/ml, preferably 4xl0 "6 to 6xl0 "6 mmol/ml.
  • eukaryotic cells can be electroporated with the method of the invention, such as vertebrate cells including mammalian cells (such as human cells, rodent (mouse, rat) cells), non-vertebrate cells (such as cells of fish and worms), lower eukaryotes such as yeasts, filamentous fungi, ascomycetes, etc.
  • mammalian cells such as human cells, rodent (mouse, rat) cells
  • non-vertebrate cells such as cells of fish and worms
  • lower eukaryotes such as yeasts, filamentous fungi, ascomycetes, etc.
  • the mammalian/human cells are preferably selected from non-hematopoietic cells including, but being not limited to, fibroblast and tumour cells, stem cells and derivatives thereof such as embryonic stem cells, hematopoietic stem cells and derivatives thereof, and hematopoietic cells including, but being not limited to, mononuclear cells, marrow CD34 + progenitor derived dendritic cells, CD34 + progenitor derived Langerhans cell, monocycle-derived dendritic cells (Mo-DC), and most preferably are Mo-DC including, but being not limited to, immature Mo- DC and mature Mo-DC, but can also be applied to DC precursors or progenitors such as monocytes or CD34+ hematopoietic progenitor cells and also to embryonic stem cells.
  • non-hematopoietic cells including, but being not limited to, fibroblast and tumour cells, stem cells and derivatives thereof such as embryonic stem cells, hematopoietic stem
  • the method of the invention is also suitable to transduce primary bone marrow cells by RNA electroporation (it could be shown that mRNA electroporation of total bone marrow mononuclear cells is possible).
  • the above mentioned precursor cells are electroporated with mRNA encoding the relevant antigen prior to (rapid) differentiation into dendritic cells. This strategy will be published in Ponsaerts et al. Journal of Immunology 2002, in press. This approach might also be of value for other types of precursor dendritic cells including CD123+ plasmacytoid dendritic cells or fresh CDllc+ blood dendritic cells that have a relative short halflife in vitro.
  • the linear polynucleotides used in embodiments (1) and (2) may be any functional nucleotide sequence exhibiting a certain effect in the eukaryotic cell, which includes polynucleotides encoding proteins or peptides to be expressed in the eukaryotic cells, polynucleotides being functional or regulatory sequences and the like.
  • the proteins or peptides to be expressed in the eukaryotic cells may or may not have a direct function in the eukaryotic cells, i.e. the expessed protein or peptide changes the property of the transfected cell, or is merely expressed in the cell or secreted by the cell (e.g. is a reporter gene or a gene product in accordance with embodiment (6)).
  • the above mentioned proteins or peptides encoded by the linear polynucleotides include, but are not limited to, tumor antigens, microbial antigens, viral antigens, immunostimulatory or tolerogenic molecules, anti-apoptotic molecules, adhesion and homing molecules and antigen processing molecules.
  • the above mentioned functional or regulatory sequences include, but are not limited to, differentiation-regulating genes, differentiation- associated genes and tissue specific genes.
  • Examples of the above proteins or peptides encoded by the linear polynucleotides are Reportergenes such as EGFP (Enhanced green fluorescent protein; SEQ ID NOs: l and 2) etc.; Tumor/Viral Antigens such as WT1 (Wilms tumor 1 protein; SEQ ID NOs:3 and 4), E6 (Human Papilloma Virus E6 protein; SEQ ID NOs:5 and 6), E7 (Human Papilloma Virus E7 protein; SEQ ID NO:7 and 8), env (Human Immunodefficiency Virus env protein; SEQ ID NO:9), gag (Human Immunodefficiency Virus gag protein SEQ ID NO: 10), tat(WT) (Human Immunodefficiency Virus tat(WT) proteins; SEQ ID NO: 11) tat(SLT) (Human Immunodefficiency Virus tat(SLT) protein SEQ ID NO: 12), Nef (Human Immunodefficiency Virus
  • RNA electroporation For the electroporation, the following parameters were most preferred: a 4 mm cuvette with 200 ⁇ l of cell suspension and we shock the cells using 300 volts and a capacitance of 150 ⁇ F (pulse time 8-10 ms). These are optimal parameters for both leukemic K562 cells and different types of DC, both progenitor- and monocyte-derived DC. In the optimization process, other parameters were also checked, e.g., by ranging the voltage and the capacitance, as well as the volume in the cuvette, resulting in shorter or longer pulse times. In summary the following parameters for efficiency and toxicity of RNA electroporation were found:
  • the common denominator for RNA electroporation is the low voltage (range 100 V-450 V), combined with a low capacitance (150 to below 300 ⁇ F) (which is in contrast to DNA settings, for which a high capacitance is required) and a low electroporation volume (200 ⁇ l) to increase cell concentration.
  • Electroporation and incubations are all performed at room temperature and cells are resuspended in serumfree buffer (e.g. IMDM, RPMI, a serum reduced buffer (e.g. Opti-MEM®) or in optimized electroporation buffer Optimix® purchased from EquiBio, UK cat n# EKIT-E1).
  • serumfree buffer e.g. IMDM, RPMI, a serum reduced buffer (e.g. Opti-MEM®) or in optimized electroporation buffer Optimix® purchased from EquiBio, UK cat n# EKIT-E1).
  • the electroporator type is Easyject Plus® (EquiBio) which only delivers exponential decay pulses.
  • a Gene Pulser II® Biorad
  • so-called "soft pulse” electroporators such as Multiporator of Eppendorf and ECM 830 of Genetronix BTX are used.
  • IVT mRNA-based electroporation is a highly efficient and simple nonviral method to gene-modify human Mo-DC, 34-DC and 34-LC with tumor antigens.
  • the technique described in this study can serve applications in DC-based tumor vaccine development and in other gene transfer protocols requiring high-level short-term transgene expression in hematopoietic cells.
  • EasyjecT PLUS D2000 model SHV (220 V; exponential decay pulses) was purchased from EquiBio Ltd. (cat # EJ-002, Action Court, Ashford Road, Ashford, Middlesex, TW15 1XB, U.K).
  • the EasyjecT PLUS is fully microprocessor controlled via a bench top remote control unit, featuring an LCD display, membrane keypad and "Smart Card” reader/recorder. Hard copies of the parameters and initiated pulse values can be taken using the EasyjecT printer (included in the EasysyjecT PLUS). This information is invaluable in confirming your experimental procedure and giving results assured information.
  • the EasyjecT PLUS includes a multitude of safety and operating detection features to enable safe operation without compromising the experimental procedure.
  • the EasyjecT is designed to deliver single or double exponential decay pulses.
  • the EasyjecT PLUS has in addition the unique "double pulse” technology. This has been beneficial in certain cases where single pulse
  • Soft pulse Multiporator ® (Eppendorf, Hamburg, Germany), exponential decay pulse and ECM830 ® CGenetronics BTX, San Diego, CA. USA ⁇ l. Rectangular pulse.
  • Electroporation cuvettes Throughout the experiments with EasyjecT PLUS D2000, sterile 4mm electroporation cuvettes with cap (EquiBio, UK cat # ECU- 104) were used. Each cuvette is individually wrapped and gamma-irradiated. Specifically designed sterile pipettes were used to further improve aseptic procedures. Total capacity is 800 ⁇ l.
  • Electroporation medium Just before electroporation, cells were resuspended in Optimix® medium (EquiBio, UK, cat #EKIT-E1). Optimix is a QC-tested fully optimised medium, designed for the electroporation of eukaryotic cells. Optimix improves both transfection efficiencies and survival rates over phosphate- buffered saline (PBS) or other standard culture medium.
  • PBS phosphate- buffered saline
  • the composition of Optimix has been carefully formulated to help protect cells during the electroporation process, also providing additional salts and critical molecules that help in the regeneration process following the destabilisation caused by the electrical discharge through the cell.
  • the Optimix kit is ready to use and contains enough material for approx. 24 experiments.
  • Optimix comprises 1 x 200ml of washing solution, 4 x 2.5ml of Optimix, 4x ATP and 4 x glutathione. Prior to use use, 5.5 mg ATP and 7.7 mg gltathione is mixed with 2.5 ml Optimix buffer and frozen in aliquots at -20°C.
  • V is the output voltage of the electroporation apparatus and d is the distance between the electrodes of the cuvette.
  • Pulse time ( ⁇ ) is by definition the elapsed time, in seconds, from the beginning of the pulse, when the electric field is maximum (E 0 ) until the electric field has decreased to e "1 (0.368) of the initial value E 0 . Practically, this value is measured by the microprocessor of the electroporation unit.
  • the pulse time gives an estimation of the duration of the membrane pore formation process and is inversely correlated by the volume of electroporation medium in the cuvette and the directly correlated with the cell concentration in the cuvette and the resistance of the medium.
  • T2 cells T2 cells (TAP-deficient, HLA-A2 + , TxB hybrid), EBV-LG2 (HLA-A2- EBV- transformed B lymphocytes), and SK23-MEL (Melan-A + HLA-A2 + melanoma cell line) were kindly provided by Dr. Pierre Van der Bruggen (Ludwig institute for Cancer Research, Brussels, Belgium).
  • K562 cells were obtained from the American Type Culture Collection (ATCC n° CCL-243, Rockville, MD, USA).
  • IMDM Iscove's medium
  • L-glutamine 2 mM
  • penicillin 100 U/ml
  • streptomycin 100 ⁇ g/ml
  • amphotericin B 1.25 ⁇ g/ml Fungizone
  • FCS 10% fetal calf serum
  • the TIL clone was maintained in AIM-V medium (Gibco BRL) supplemented with 5% pooled human AB serum (Sigma, Bornem, Belgium) and 500 IU/ml interleukin (IL)-2 (R&D Systems, Minneapolis, MN, USA) and used as responder population in DC coculture experiments.
  • AIM-V medium Gibco BRL
  • human AB serum Sigma, Bornem, Belgium
  • 500 IU/ml interleukin (IL)-2 R&D Systems, Minneapolis, MN, USA
  • BM samples were aspirated by sternal puncture from hematologically normal patients undergoing cardiac surgery, after informed consent.
  • Peripheral blood mononuclear cells PBMC
  • the 6 PBMC donors used in this study are designated by letters A to F.
  • Mononuclear cells were isolated by Ficoll- Hypaque gradient separation (LSM, ICN Biomedicals Inc., Costa Mesa, CA, USA). Monocytes were directly isolated and used for DC culture, as described below.
  • PBMC for DC/T-cell cocultures were cryopreserved in a solution consisting of 90% FCS and 10% DMSO and stored at -80°C until use.
  • CD34 + cell sorting After Ficoll-Hypaque separation, mononuclear BM cells were indirectly stained using supernatant of the 43A1 hybridoma (anti-CD34) kindly donated by Dr. H-J. B ⁇ hring, University of Tubingen, Germany (Buhring, H. J. et al., Leukemia, 5:854-860 (1991)), followed by fluorescein isothiocyanate (FITC)- conjugated rabbit anti-mouse immunoglobulins (DAKO, Glostrup, Denmark).
  • anti-CD34 43A1 hybridoma
  • the CD34 labeled cells were then sorted on a FACStar PLUS cell sorter (Becton Dickinson, Erembodegem, Belgium) equipped with an air-cooled argon ion laser ILT model 5500-A (Ion Laser Technology, Salt Lake City, UT, USA). Sort windows were set to include cells with low side scatter and with positive green fluorescence (CD34 + ). Purities of >95% were routinely obtained.
  • CD34 + cells were cultured in 2 ml of complete medium supplemented with 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; Leucomax, Novartis Pharma, Basel, Switzerland), 2.5 ng/ml tumor necrosis factor (TNF)- ⁇ (Roche Molecular Biochemicals, Mannheim, Germany) and 50 ng/ml stem cell factor (SCF; Biosource, Nivelle, Belgium) until day 5; afterwards, SCF was replaced by 1000 U/ml IL-4 (R&D Systems), which was added for the next 5-9 days. After 12 days of culture, a 15-20 fold total cell expansion was observed and cells exhibited typical markers of mature DC including CDla, CD80, CD86 and HLA-DR (Fig. 3C).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • TNF tumor necrosis factor
  • SCF stem cell factor
  • sorted CD34 + cells were first cultured for 8 days in complete medium containing 100 ng/ml IL-3, 100 ng/ml IL-6 and 50 ng/ml SCF (all from Biosource), followed by LC differentiation in GM-CSF (100 ng/ml) and IL-4 (1000 U/ml) for the next 4 weeks.
  • Immature monocyte-derived DC were generated from PBMC as described by Romani, N. et al., J. Exp. Med., 180:83-93 (1996). Briefly, PBMC were allowed to adhere in AIM-V medium for 2 h at 37°C. The non-adherent fraction was removed, and adherent cells were further cultured for 5-7 days in IMDM supplemented with 2.5% autologous heat-inactivated plasma. GM-CSF (100 ng/ml) and IL-4 (1000 U/ml) were added to the cultures every 2-3 days starting from day 0. Maturation of iMo-DC was induced by adding 2.5 ng/ml TNF- ⁇ and 100 ng/ml lipopolysaccharide (LPS; Sigma) for 24 h starting from day 6 of the Mo-DC culture.
  • LPS lipopolysaccharide
  • monocytes derived from PBMCs were allowed to adhere in AIM-V medium (Gibco BRL, Paisly, UK) for 2 h at 37°C in 6-well culture plates (20xl0 6 PBMC/well). After careful removal of the non-adherent fraction, cells were cultured in serum-free AIM-V medium supplemented with 100 ng/ml GM-CSF (Leucomax, Novartis Pharma, Basel, Switzerland) for 2 days. To obtain mature DC, poly-I:C (Sigma, Cambridge, UK) was added 24 hours after starting the culture at a concentration of 25 ⁇ g/ml. The typical yield and purity of the DC culture was l-2xl0 6 cells/well containing 60-70% of DC.
  • monocytes were isolated from PBMC by magnetic isolation using CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer's instructions. Routinely, 4-8xl0 6 monocytes were obtained starting from lOOxlO 6 PBMC with purity levels > 85%.
  • HLA-A typing of DC HLA-A2 subtyping was determined on BM-derived mononuclear cells and PBMC by indirect staining with the supernatant of the BB7-2 hybridoma (anti-HLA-A2; ATCC), followed by FITC-conjugated rabbit anti- mouse immunoglobulins (DAKO). HLA-A2 staining was analyzed by flow cytometry using a FACScan analytical flow cytometer (Becton Dickinson, Erembodegem, Belgium).
  • Synthetic peptides An influenza virus-specific HLA-A*0201-restricted matrix protein Ml peptide (Ml; amino acids (aa) 58-66, GILGFVFTL; SEQ ID NO:32) was used for activation or for detection of matrix protein Ml peptide specific T- cells when pulsed on respectively DC and T2 cells.
  • Ml matrix protein Ml peptide
  • Melan-A peptide (MA; aa 27-35, AAGIGILTV; SEQ ID NO:34) was also used.
  • Peptides (>95% pure) were purchased from Sigma-Genosys (Cambridge, UK). Both peptides were dissolved in 100% DMSO to 10 mg/ml, further diluted to 1 mg/ml in serum-free IMDM and stored in aliquots at -70°C. Peptides were used at a final concentration of 20 mM. The peptides (>95% pure) were purchased from Sigma-Genosys (Cambridge, UK).
  • the peptides were dissolved in 100% DMSO to 10 mg/ml, further diluted to 1 mg/ml in serum-free IMDM and stored in aliquots at -80°C. The peptides were used at a final concentration of 20 ⁇ M.
  • T2 cells, HLA-A2 + iMo-DC or DC were washed twice with IMDM and subsequently incubated (2xl0 6 cells/ml) for 1 to 2 h at room temperature in 5 ml conical polystyrene tubes or 15 ml conical tubes with 20 ⁇ g/ml peptide in serum-free IMDM medium supplemented with 2.5 ⁇ g/ml ⁇ 2- microglobulin (Sigma). Afterwards, the cells were washed and used respectively as stimulators for PBMC or as restimulators in cytokine release assays.
  • Plasmids For plasmid cDNA transfection, a pEGFP-Nl plasmid (CLONTECH Laboratories, Palo Alto, CA, USA) was used encoding an enhanced green fluorescent protein (EGFP) gene under the control of a CMV promoter plasmid pGEM4Z/EGFP/A64 (kindly provided by Dr. E. Gilboa, Duke University Medical Center, Durham, NC, USA) contained the EGFP gene under the control of T7 promoter. Plasmid pcDNAl.l/Melan-A contained the Melan-A/MART-1 gene driven by a CMV promoter was kindly provided by Dr. Pierre Van der Bruggen.
  • EGFP enhanced green fluorescent protein
  • Plasmid pcDNAl.l/Amp (Invitrogen, Carlsbad, CA, USA) was used as a backbone control vector.
  • Plasmid pCMV-Luciferase (CLONTECH Laboratories, Polo Alter, CA, USA) carried a luciferase gene under the control of a CMV promoter and was used as a control plasmid.
  • Plasmid pGEM4Z/Ml/A64 (kindly provided by Dr. A. Steinkasserer, University of Er Weg, Er Weg, Germany) encoding an influenza Ml gene under the control of a T7 promoter (SEQ ID NO: 31). Plasmids were propagated in E.
  • Coli strain DH5 ⁇ (Gibco BRL) or supercompetent cells (Stratagene, La Jolla, CA, USA) and purified on endotoxin-free QIAGEN®-tip 500 columns (Qiagen, Chatsworth, CA, USA).
  • plasmids were linearized, purified using a Genieprep kit (Ambion, Austin, TX, USA) or a PCR purification Kit (Qiagen) and used as DNA templates for the in vitro transcription reaction.
  • pcDNAl.l/Melan-A was used as such for in vitro transcription under the control of a T7 promoter.
  • EGFP cDNA isolated as a 0.8 kb Hindlll-Notl fragment from pEGFP-Hl, was first subcloned into pcDNAl.l/Amp and subsequently cloned as a BamHI-Xbal fragment into pSP64 (Promega, Madison, WI, USA) that allows in vitro transcription under the control of an SP6 promoter. Transcription was carried out in a final 20-100 ⁇ l reaction mix at 37°C for 3-4 h using the SP6 MessageMachine kit (Ambion) to generate 5' m 7 GpppG- capped IVT mRNA. Transcription reactions with Spe I (MBI Fermentas, St.
  • CD14 microbead-isolated monocytes were washed twice with Optimix Washing Solution (EquiBio, Ashford, Middlesex, UK) and resuspended to a final concentration of 50xl0 6 cells/ml in Optimix electroporation buffer (EquiBio). Subsequently 0.2 ml of the cell suspension was mixed with 20 ⁇ g of IVT mRNA and electroporated in a 0.4 cm cuvette at 300 V and 150 ⁇ F using an Easyject Plus device (EquiBio). Plasmid DNA electroporation was performed as previously described (Van Tendeloo, V.F.I, et al., Gene Ther., 5:700-707 (1998)). After electroporation, fresh complete medium (including cytokines for DC) was added to the cell suspension and cells were further incubated at 37°C in a humidified atmosphere supplemented with 5% CO 2 .
  • Lipofection of mRNA was performed using the cationic lipid DMRIE-C (Gibco BRL) according to manufacturer's instructions with minor modifications (Van Tendeloo, V.F.I, et al., Gene Ther., 5:700-707 (1998)). Briefly, K562 cells were washed twice with serum-free IMDM and resuspended to a final concentration of 1-2.10 6 cells/ml in Opti-MEM. 34-DC, 34-LC and Mo-DC were harvested after respectively 12, 25 and 6 days of culture, washed twice with serum-free IMDM, and resuspended to a final concentration of 1-2.10 6 cells/ml in Opti-MEM.
  • IVT mRNA Five ⁇ g of IVT mRNA, diluted in 250 ⁇ l Opti-MEM, was mixed with DMRIE-C, also diluted in 250 ⁇ l Opti-MEM, at a lipid:RNA ratio of 4: 1. After 5-15 min of incubation at room temperature in order to allow RNA-lipid complexatfon, lipoplexes were added to the cells and allowed to incubate for 2 hours at 37°C. Alternatively, 5-20 ⁇ g of IVT mRNA was pulsed to the cells in the absence of DMRIE-C for 3-4 h at 37°C. Plasmid DNA lipofection was performed as described previously (Van Tendeloo, V.F.I, et al., Gene Ther., 5:700-707 (1998)). After lipofection or passive pulsing, fresh complete medium (including cytokines for DC) was added to each well.
  • EGFP analysis EGFP-transfected cells were checked for EGFP expression 24-48 h after transfection by flow cytometric (FCM) analysis. Briefly, cells (1-5 x 10 5 ) were washed once in phosphate-buffered saline (PBS) supplemented with 1% FCS and resuspended in 0.5 ml of PBS supplemented with 1% BSA and 0.1% sodium azide. Ethidium bromide (EB) at a final concentration of 10 ⁇ g/ml was added directly prior to FCM analysis on a FACScan analytical flow cytometer (Becton Dickinson) to assess cell viability.
  • FCM flow cytometric
  • gating was performed on cells exhibiting a large forward scatter (FSC) and side scatter (SSC) profile, i.e. DC, in order to allow exclusion of contaminating autologous lymphocytes. Gated DC were then evaluated for EGFP expression.
  • FSC forward scatter
  • SSC side scatter
  • Immunophenotyping of DC Immunophenotyping was performed as described previously (Van Tendeloo, V.F.I, et al., Gene Ther., 5:700-707 (1998)). The following monoclonal antibodies were used: CDla-fluorescein isothiocyanate (FITC) (Ortho Diagnostic Systems, Beerse, Belgium), CDla-phycoerythrin (PE) (Caltag Laboratories, San Francisco, CA, USA), CD14-PE, HLA-DR-PE, HLA-DR- FITC (PharMingen, San Diego, CA, USA), CD4-PE, CD80-PE (Becton Dickinson), CD80-FITC (PharMingen, San Diego, CA, USA), CD40-FITC (BioSource, Zoersel, Belgium), CD86-PE (PharMingen, San Diego, CA, USA), CD86-FITC (Serotec, Oxford, UK), CD13-FITC (DAKO), CD14-FITC (Be
  • Immunophenotyping with CD83 was followed by staining with a secondary rabbit anti-mouse (RAM)-FITC antibody (Dako, Glostrup, Denmark).
  • RAM rabbit anti-mouse
  • Nonreactive isotype-matched antibodies (Becton Dickinson) were used as controls.
  • Ethidium bromide was added prior to FCM analysis on a FACScan analytical flow cytometer (Beckton Dickinson) to assess cell viability and to exclude dead cells from the analysis. Gating was also performed to exclude remaining lymphocytes in the DC cultures.
  • EGFP enhanced green fluorescent protein
  • PE phycoerythrin
  • Interferon (IFN)- ⁇ release assay 34-DC, 34-LC and iMo-DC were used as stimulator cells 24 h after transfection.
  • IFN Interferon
  • 6-day- cultured iMo-DC were allowed to mature for 24 h in the presence of TNF- ⁇ and LPS prior to transfection and used as stimulators 24 h after transfection.
  • iMo-DC were transfected with mRNA on day 6 of culture and, after 12-16 h to allow protein expression, TNF- ⁇ and LPS were added to induce final DC maturation. After an additional 24 h, mature transfected Mo-DC were used as stimulators.
  • iMo-DC pulsed with the Melan-A, an irrelevant influenza Ml peptide or an irrelevant human papilloma virus E7 peptide were used as stimulators.
  • Stimulators were either washed twice and resuspended in AIM-V medium supplemented with 10% pooled human AB serum and 40 IU/ml IL-2.
  • Responder CTL were washed vigorously 3-4 times and resuspended in AIM- V medium. Then, CTL (1 x 10 5 cells) were coincubated with stimulator cells (lxlO 5 cells) in 96-round bottom plates for 24 h at 37°C in a total volume of 200 ⁇ l.
  • stimulators and responder PBMC were washed and resuspended in IMDM + 5% hAB serum. Then, responder PBMC (lxlO 5 cells) were coincubated with stimulator cells (lxlO 4 cells) in 96-well round-bottom plates for 6 hours at 37°C in a total volume of 100 ⁇ l. Triplicate supernatant samples from these cocultures were tested for specific IFN- ⁇ secretion by an IFN- ⁇ ELISA (Biosource, Nivelle, Belgium). To normalize data, the background IFN- ⁇ secretion (defined as IFN- ⁇ released by the CTL exposed to unmodified DC) was subtracted from each of the observed measurements. Measurements are presented as IU/ml released by 10 5 responder cells/24 h.
  • IFN- ⁇ secreting cell assay PBMC primed and cultured as described above (lxlO 6 ) were restimulated for 3 hours in 24-well plates with T2 cells (lxlO 5 ) pulsed with Ml peptide or E7 peptide as control. Next, IFN- ⁇ -secreting cells were analyzed by a flow cytometric IFN- ⁇ Secretion Assay Detection Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer's instructions. Cells were also stained with CD8-FITC (Becton Dickinson) and 5xl0 5 cells were analyzed per sample by flow cytometry. Analysis was done by gating on the lymphocyte population.
  • Allogeneic mixed leukocyte reaction Immature and mature DC were used for stimulation of allogeneic PBMC. Briefly, immature or mature DC were cocultured with 20xl0 6 allogeneic PBMC (ratio 1: 10) in 10 ml IMDM supplemented with 5% human (h) AB serum (Sigma) in T25 culture flasks. On day 4 of culture, 5 ml fresh medium (IMDM + 5% hAB serum) was added to the cultures. On day 7 of culture, cells were analyzed for reactivity.
  • MLR mixed leukocyte reaction
  • stimulated PBMC (lxlO 5 cells) were restimulated with PBMC from the DC donor (lxlO 4 cells) in 96-well round bottom plates for 6 hours at 37°C in a total volume of 100 ⁇ l.
  • Supernatant samples from these cocultures were tested for IFN- ⁇ secretion by IFN- ⁇ ELISA (Biosource, Nivelle, Belgium).
  • Induction of MHC class 1-restricted influenza-specific T cells Ml peptide-pulsed immature, Ml peptide-pulsed mature DC and matrix protein mRNA- electroporated mature DC were used for antigen-specific stimulation of PBMC.
  • 2xl0 6 antigen-loaded DC were cocultured with 20xl0 6 autologous PBMC (ratio 1: 10) in 10 ml IMDM supplemented with 5% hAB serum in T25 culture flasks.
  • 10 ml IMDM supplemented with 5% hAB serum was added to the cultures.
  • 5 ml fresh medium was added to the cultures.
  • cells were analyzed for antigen specificity.
  • mRNA electroporation at optimal settings showed a significantly reduced cell mortality rate as compared to cDNA electroporation at optimal settings (15% versus 51%, respectively).
  • DMRIE-C-mediated RNA and DNA lipofection showed a somewhat similar outcome in terms of efficiency and viability although optimal lipid:nucleic acid ratio (4:1 versus 3:1) as well as incubation time (2 h versus 6 h) varied for RNA and DNA lipofection, respectively (Table 1).
  • RNA is extremely labile and has a short half-life time compared to DNA
  • Fig. IB we also studied kinetics of EGFP expression following mRNA electroporation
  • Immature Mo-DC Immature Mo-DC (iMo-DC) were generated from adherent PBMC in the presence of GM-CSF and IL-4. At day 5-6 of culture, Mo-DC were electroporated with EGFP mRNA. Optimization experiments revealed optimal settings similar to those of K562 cells (300 V, 150 ⁇ F), leading to maximal transfection efficiency combined with the lowest level of cell death. FCM analysis of EGFP expression showed more than 60% EGFP-expressing iMo-DC (Fig. 2A & Table 2).
  • mRNA transfection in bone marrow CD34 + progenitor-derived DC (34-DC) and CD34 + progenitor-derived Langerhans cells (34-LC) was also tested. Up to 72% and 53%, respectively, of these DC types were readily transfected by mRNA electroporation (Fig. 2C), but not by mRNA lipofection or mRNA pulsing (Table 2). Viability was always higher than 80% for both 34-DC and 34-LC (Fig. 2C). Table 2 summarizes efficiency of mRNA-based electroporation, lipofection and passive pulsing in the different types of DC.
  • EGFP expression was analyzed by FCM to estimate transfection efficiency (% EGFP + DC).
  • iMo-DC immature Mo-DC
  • mMo-DC mature Mo-DC
  • 34-LC CD34 + progenitor-derived Langerhans cells
  • 34-DC CD34 + progenitor-derived dendritic cells.
  • SD standard deviation
  • Phenotype and maturation of mRNA-electroporated DC Since DC have a delicate phenotype which can easily be disturbed by culture or transfection conditions, we assessed by FCM analysis whether electroporated DC retained their respective phenotype as well as their capacity to differentiate into mature DC. Control and EGFP mRNA-transfected Mo-DC were stained using monoclonal antibodies binding to characteristic DC markers including CDla, HLA-DR, CD80, CD86 and CD83. Electroporation of mRNA showed no effect on the phenotype of Mo-DC, as electroporated Mo-DC co-expressing EGFP retained high levels of CDla, HLA-DR and CD86 (Fig. 3A).
  • EGFP + 34-DC co-expressed HLA-DR, CDla, CD80 and CD86. Similar findings were observed in 34-LC, with the exception that 34-LC exhibited lower levels of CD80 and CD86, compatible with their similarity to immature Langerhans-like DC (data not shown).
  • MHC class I-restricted antigen presentation by mRNA-transfected DC Given the high transfection efficiency in Mo-DC, we investigated to what extent mRNA- transfected Mo-DC could process antigen and present MHC class I-restricted antigenic epitopes to an antigen-specific CTL clone. Therefore, we introduced mRNA encoding Melan-A/MART-1 into HLA-A2 + Mo-DC using electroporation, lipofection or passive pulsing. Mo-DC electroporated or lipofected with Melan-A mRNA markedly stimulated an HLA-A2 + Melan-A-specific CTL clone, as judged by IFN- ⁇ secretion (Fig. 4).
  • Mo-DC passively pulsed with Melan-A mRNA did not result in any CTL stimulation.
  • HLA-A2 + Mo-DC electroporated with EGFP mRNA or HLA-A2 ' Mo-DC electroporated with Melan-A mRNA did not stimulate the CTL clone to produce IFN- ⁇ .
  • iMo-DC immature Mo-DC
  • mMo-DC mature Mo-DC
  • 34-LC CD34 + progenitor-derived Langerhans cells
  • 34-DC CD34 + progenitor-derived DC
  • ⁇ BG IFN- ⁇ production below background.
  • Mo-DC obtained by culturing PBMC in the presence of GM-CSF and IL-4 for 5-7 days exhibit predominantly an immature phenotype (Romani, N. et al., J. Immunol. Methods, 196: 137-151 (1996)). These immature Mo-DC are specialized in capturing large amounts of antigens from the environment (Sallusto, F., Lanzavecchia, A., J. Exp. Med., 179: 1109-1118 (1994)). However, for optimal presentation to CTL, Mo-DC need to undergo a maturation process which can be induced by bacterial products (e.g.
  • 34-LC and 34-DC can also be transfected by plasmid DNA electroporation (Van Tendeloo, V.F.I, et al., Gene Ther., 5:700-707 (1998)). Therefore, we evaluated whether plasmid DNA-transfected DC can also induce antigen-specific CTL activation.
  • HLA-A2 + 34- LC electoporated with plasmid DNA or IVT mRNA encoding Melan-A were incubated with the Melan-A specific CTL to evaluate IFN- ⁇ secretion (Fig. 6).
  • EGFP RNA-transfection of immature monocyte-derived dendritic cells (generated from leukapheresis products and matured by a cocktail of IL-l ⁇ + IL-6 + TNF ⁇ + PEG 2 under GMP conditions for clinical application) by electroporation.
  • Monocyte- derived immature Dendritic Cells (DC) were generated from leukapheresis products as described (Feuerstein, B. et al., J. Immunol, Methods 245: 15-29 (2000)).
  • Immature DC (d6) were washed twice in RPMI and once in washing- solution of the Optimix ® -Kit (EQUIBIO, Maidstone Kent, U.K.).
  • DC were adjusted to a final cell concentration of 10xl0 6 /ml in Optimix ® -Medium. Then 0,2 ml of the cell suspension were mixed with 20 ⁇ g in vitro transcribed EGFP RNA in a 1,5 ml reaction tube. After incubation at room temperature for a maximum of 3 minutes the cell suspension were transferred in a 0,4-cm-gap electroporation-cuvette. Pulse were triggered at a voltage of 300 V and a capacitance of 150 ⁇ F with the Gene Pulser II (BioRad, Kunststoff, Germany) resulting in pulse time of 7-10 msec.
  • DC Dendritic Cells
  • Immature DC (d6) were washed twice in RPMI and once in washing-solution of the Optimix ® -Kit (EQUIBIO, Maidstone Kent, U.K.). DC were adjusted to a final cell concentration of 10xl0 6 /ml in Optimix ® -Medium. Then 0,2 ml of the cell suspension were mixed with or without 20 ⁇ g in vitro transcribed
  • EGFP RNA in a 1,5 ml reaction tube. After incubation at room temperature for a maximum of 3 minutes the cell suspension were transferred in a 0,4-cm-gap electroporation-cuvette. Pulse were triggered at the indicated voltage and a capacitance of 150 ⁇ F with the Gene Pulser II (BioRad, Kunststoff, Germany) resulting in pulse time of 7-10 ms. Immediately after that the cell suspensions were transferred to 6-well-plates (lxlO 6 DC/ well/3 ml culture medium). Terminal maturation was induced by addition of IL-l ⁇ , IL-6, TNF-a and PGE 2 as described (Feuerstein, B. et al., J. Immunol. Methods 245: 15-29 (2000)). 48 h after electroporation the DC were analyzed. The contour-plots of Fig. 9A show on the x-axis the forward side scatter and on y-axis the sideward scatter.
  • the Forward and Side Scatter analysis addition reveals that for monocyte-derived Dendritic Cells that are generated from leukapheresis products, RNA-transfected by electroporation, and fully matured by adding a maturation cocktail consisting of of IL-l ⁇ , IL-6, TNF-a and PGE 2 (Feuerstein, B. et al., J. Immunol. Methods, 245: 15-29 (2000)) the use of 260 V is slightly better as the integrity of the cells is somewhat better preserved.
  • Immature DC ⁇ ) - see Fig. 9A - were washed twice in RPMI and once in washing-solution of the Optimix ® -Kit (EQUIBIO, Maidstone Kent, U.K.). DC were adjusted to a final cell concentration of 10xl0 6 /ml in Optimix ® -Medium. Then 0,2 ml of the cell suspension were mixed with or without 20 ⁇ g in vitro transcribed EGFP RNA in a 1,5 ml reaction tube. After incubation at room temperature for a maximum of 3 minutes the cell suspension was transferred in a 0,4-cm-gap electroporation-cuvette.
  • Pulses were triggered at the indicated voltage and a capacitance of 150 ⁇ F with the Gene Pulser II (BioRad, Kunststoff, Germany) resulting in pulse time of 7-10 msec.
  • the cell suspensions were transferred to 6-well-plates (lxlO 6 DC/ well/3 ml culture medium). Terminal maturation was induced by addition of IL-l ⁇ , IL-6, TNF-a and PGE 2 .
  • the DC were counterstained with the indicated mouse mAbs and PE-conjugated anti-mouse Ig followed by FACS-analysis. The results are shown in Fig. 9B.
  • the phenotypic analysis reveals that for monocyte-derived Dendritic Cells that are generated from leukapheresis products, RNA-transfected by electroporation, and fully matured by adding a maturation cocktail consisting of of IL-l ⁇ , IL-6, TNF-a and PGE 2 (Feuerstein, B. et al., J. Immunol. Methods, 245: 15-29 (2000)) the use of 260 V is slightly better as more cells are in the upper right quadrant, i.e. expressing both EGFP and the maturation markers CD83 and CD25.
  • EGFP RNA-transfection of already matured monocyte-derived dendritic cells (generated from leukapheresis cells and matured by a cocktail of IL-l ⁇ + IL-6 + TNF ⁇ + PEG 2 under GMP conditions for clinical application) by electroporation.
  • Immature DC (d6) were induced to undergo terminal maturation by addition of IL-l ⁇ , IL-6, TNF- ⁇ and PGE 2 as described in Feuerstein,. B. et al., J. Immunol. Methods 245: 15-29 (2000).
  • Mature DC were transfected with EGFP-RNA by electroporation as described in Example 2.
  • DC Bi Mature monocyte-derived Dendritic Cells
  • K562 cells were electroporated with EGFP mRNA and cryopreserved 3 or 24 hours after transfection.
  • K562 cells were resuspended in cryotubes (Nunc CryoTube Vials, Nalgene Nunc International, Denmark) at a concentration of 10 x 10 6 per ml in pure FCS.
  • the suspension was mixed on ice with an equal volume of FCS supplemented with 20% DMSO (Sigma, St. Louis, MO, USA).
  • Cell suspensions were slowly frozen (-l°C/min) to -80°C by using a cryo freezing container (Nalgene Nunc International). Cells were frozen at -80°C for more than 24 hours before use in further experiments.
  • Immature Mo-DC were electroporated with EGFP mRNA.
  • Cells were cryopreserved as immature DC 18 hours after transfection or as mature DC 24 hours after transfection. Maturation was induced by adding a maturation cocktail (TNF- ⁇ + PGE 2 + IL-1 + IL-6) directly after transfection.
  • FCM maturation cocktail
  • DC were frozen 24 hours after mRNA electroporation and transgene expression and cell survival was determined 6 and 24 hours after thawing (Table 2 ; Figure 1 B).
  • thawing Six hours after thawing, DC cultures appeared to survive the freezing and have a similar number of EGFP+ cells and MFI level of EGFP+ cells as compared to non-frozen cultures (p-value respectively 0.0033 and 0.5183).
  • a combination of a serum-free culture protocol and a poly-I:C maturation stimulus results in the rapid generation of fully mature and viable CD83+ DC from peripheral blood monocytes.
  • This provides for an efficient and clinical applicable antigen loading strategy for these short-term cultured DC, based on mRNA electroporation of monocytes.
  • the T-cell activation capacity of these short-term and serum-free cultured Mo-DC was found to be highly stimulatory in an influenza antigen model system using influenza matrix protein Ml peptide- pulsed and matrix protein mRNA-electroporated DC. In the following (including the corresponding Figures) results are expressed as mean ⁇ standard deviation. Comparisons were validated using Student's t-test. A p-value ⁇ 0.05 was considered to be statistically significant.
  • a ⁇ Characterization of short-term and serum-free in vitro cultured DC, with or without poly-I:C maturation After monocyte enrichment from PBMC, cells were cultured for 2 days in AIM-V medium supplemented with GM-CSF only. To obtain mature DC, poly-I:C was added after 24 hours of culture. Cultured cells were analyzed after a total culture period of 48 hours by flow cytometry. One observed difference with classical DC cultured for 6-7 days in serum-containing medium supplemented with GM-CSF and IL-4, was a lower forward- and side-scatter profile of the serum-free-cultured cells (Figure 1, upper panels).
  • J Poly-I:C maturated serum-free-cultured DC are more potent than their immature counterparts in inducing in vitro T-cell immune responses:
  • their stimulatory capacity was first evaluated in a modified allogeneic mixed leucocyte reaction (MLR).
  • MLR modified allogeneic mixed leucocyte reaction
  • immature and mature DC were cultured for 7 days with allogeneic PBMC.
  • the stimulated PBMC were restimulated with PBMC from the DC donor, and IFN- ⁇ secretion in the supernatant was analyzed by ELISA (Figure 16).
  • mRNA electroporation of monocytes followed by differentiation to DC Using a previously optimized mRNA electroporation protocol, we examined the possibility of genetic modification of the above-described DC. In these experiments, the EGFP reporter gene was used to assess mRNA transfection efficiency. After optimization, the following mRNA electroporation and culture protocol resulted in the generation of antigen-loaded mature DC.
  • monocytes were isolated from PBMC by CD14 immunobead magnetic separation. After electroporation, cells were resuspended in serum-free AIM-V medium supplemented with GM-CSF. After 24 hours of culture, poly-I:C was added to the cultures to obtain mature DC. The cultured DC were analyzed 48 hours after electroporation of the monocytes.
  • the phenotype of the cultured cells was examined by flow cytometry for the characteristic DC markers (Figure 20).
  • Figure 20 We observed no difference in phenotype between non-electroporated and EGFP mRNA-electroporated short-term and serum-free cultured mature DC.
  • D. Stimulatory capacity of mRNA-loaded short-term-cultured mature DC We examined in an influenza model system whether mRNA-electroporated monocytes rapidly differentiated in serum-free medium into mature DC could stimulate antigen-specific T-cells upon coculture with PBMC. In these experiments, monocytes were electroporated with mRNA encoding influenza matrix protein Ml, and further cultured to mature DC as described above. Next, DC were cocultured with autologous PBMC without the addition of exogenous cytokines.
  • primed PBMC were restimulated with T2 cells pulsed with a MHC class I-restricted influenza matrix protein Ml peptide (T2/M1), and IFN- ⁇ secretion was determined after 6 hours by ELISA ( Figure 21).
  • T2 cells pulsed with a MHC class I-restricted influenza matrix protein Ml peptide (T2/M1)
  • IFN- ⁇ secretion was determined after 6 hours by ELISA ( Figure 21).
  • the activated T cells in the primed PBMC culture produced IFN- ⁇ against the immunodominant Ml matrix protein peptide.
  • type I interferons like IFN- ⁇ , might induce IL-15 production and in this way strongly promote a T-helper 1 response, which is needed for induction of a strong CD8+ T cell response (Santini, S.M. et al. 2000. Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in Hu-PBL-SCID mice. J. Exp. Med. 10:1777; Saikh, K.U., et al. 2001.
  • mRNA- electroporated mature dendritic cells retain transgene expression, phenotypical properties and stimulatory capacity after cryopreservation.
  • Leukemia in press
  • the level of protein expression e.g. EGFP
  • Monocytes that were electroporated with EGFP-mRNA and subsequently differentiated to mature DC showed only a small shift of EGFP fluorescence as compared to non-electroporated control DC. This can be explained by the difficulty of obtaining high protein expression levels in primary uncultured mononuclear cells (data not shown).
  • Example 7 mRNA electroporation of adult bone marrow.
  • EP parameters 300V, 150 ⁇ F (mRNA settings) or 260V, 1050 ⁇ F (DNA settings)
  • This experiment shows that the mRNA electroporation technology of the present invention is able to transfect human bone marrow mononuclear cells up to 25- 30% efficiency.
  • High levels of EGFP expression were observed in the myeloid fraction (CD33+ cells), in particular in the monocyte fraction (CD14+ cells) and the hematopoietic progenitor fraction (CD34+ cells) comprising the hematopoietic stem cells.
  • a low but consistent transfection level was observed in the lymphoid fraction (CD7+ and CD19+ cells), concordant with the data obtained in peripheral blood (see Fig. 22A to E).
  • Example 8 mRNA electroporation of mouse embryonic stem cells.
  • EP parameters 300V, 150 ⁇ F (mRNA settings)
  • ES cells 5 million ES cells were thawed on 10/6 and put into culture in gelatin-coated 75 cm 2 flask and 3 million mitomycin C-treated mouse embryonic fibroblasts (MEF) feeder cells for 48h. Then, ES cells were trypsinized, washed 3 times in DMEM, once in Electroporation Wash Buffer and resuspended in Optimix medium at 7.5 million cells/200 ⁇ l. 20 ⁇ g of EGFP mRNA or 20 ⁇ l RNase-free water (mock) was added to the cells just before electroporation.
  • MEF mouse embryonic fibroblasts
  • EGFP fluorescence was checked by fluorescence microscopy at 24h post-EP and simultaneous EGFP and phenotypic analysis was performed at 48h post-EP by FACS (results see Fig. 23A and B).
  • the mRNA electroporation technology of the present invention is able to transfect mouse embryonic stem (ES) cells to levels above 90% efficiency, implicating a powerful tool to genetically modify mouse ES cells, and possibly also human ES cells, be it in a transient manner. This could be of value for control of differentiation of ES cells by transgene expression of master regulator genes, skewing or biasing differentiation into distinct lineages for large-scale generation of differentiated cells and tissues in vitro.
  • ES mouse embryonic stem
  • EP parameters 300V, 150 ⁇ F (mRNA settings)
  • Fresh PBMC were washed twice in IMDM, once in Electroporation Wash Buffer and resuspended in Optimix medium at 5 million cells/200 ⁇ l.
  • EGFP mRNA or 20 ⁇ l RNase-free water was added to the cells just before electroporation. After shocking, cells were immediately put into 3 ml of warm culture medium (IMDM, 10% FCS). Simultaneous EGFP and phenotypic analysis was performed at 24h by FACS (results see Fig. 24A-C).
  • Electroporation of mRNA at ⁇ s-range (soft pulse; general method): Immature (d6) or mature (d7) Mo-DC were washed once with Opti-Mem® or with washing- solution Optimix®, respectively.
  • Cells were adjusted to a final cell concentration of l-4xl0 7 /ml in electroporation buffer (Opti-Mem ® , Optimix ® or isoosomolar electroporation buffer). Then 0,2-0,8 ml of the cell suspension were mixed with IVT mRNA (up to 20 ⁇ g /2xl0 6 cells) in a 1,5 ml reaction tube.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Materials For Medical Uses (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une technique améliorée d'apport de gène dans des cellules eucaryotes par électroporation, de préférence dans des cellules hématopoïétiques humaines, en particulier des cellules dendritiques. La technique de cette invention est supérieure à la lipofection et à l'impulsion passive d'ARNm et à l'électroporation d'ADNc de plasmide destiné à l'apport de gène, le chargement d'antigène tumoral de cellules dendritiques y compris.
PCT/EP2002/006897 2001-06-21 2002-06-21 Transfection amelioree de cellules eucaryotes avec polynucleotides lineaires par electroporation Ceased WO2003000907A2 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
JP2003507290A JP2004536598A (ja) 2001-06-21 2002-06-21 電気穿孔による真核細胞への線形ポリヌクレオチドの改良された形質移入
CN028163621A CN1701121B (zh) 2001-06-21 2002-06-21 通过电穿孔用线性多核苷酸改善对真核细胞的转染
KR10-2003-7016741A KR20040025690A (ko) 2001-06-21 2002-06-21 진핵세포를 전기천공에 의하여 선형 폴리뉴크레오티드로형질감염시키는 개선된 형질감염 방법
EP02748810.5A EP1397500B1 (fr) 2001-06-21 2002-06-21 Transfection amelioree de cellules eucaryotes avec polynucleotides lineaires par electroporation
BR0210936-0A BR0210936A (pt) 2001-06-21 2002-06-21 Transfecção melhorada de células eucarióticas com polinucleotìdeos lineares por eletroporação
CA2451389A CA2451389C (fr) 2001-06-21 2002-06-21 Transfection amelioree de cellules eucaryotes avec polynucleotides lineaires par electroporation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP01115076A EP1270732A1 (fr) 2001-06-21 2001-06-21 Transfection de cellules eucaryotes avec des polynucléotides linéaires par électroporation
EP01115076.0 2001-06-21

Publications (2)

Publication Number Publication Date
WO2003000907A2 true WO2003000907A2 (fr) 2003-01-03
WO2003000907A3 WO2003000907A3 (fr) 2003-09-25

Family

ID=8177780

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2002/006897 Ceased WO2003000907A2 (fr) 2001-06-21 2002-06-21 Transfection amelioree de cellules eucaryotes avec polynucleotides lineaires par electroporation

Country Status (8)

Country Link
EP (3) EP1270732A1 (fr)
JP (2) JP2004536598A (fr)
KR (1) KR20040025690A (fr)
CN (1) CN1701121B (fr)
AU (2) AU2008202507B2 (fr)
BR (1) BR0210936A (fr)
CA (1) CA2451389C (fr)
WO (1) WO2003000907A2 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007090647A1 (fr) * 2006-02-08 2007-08-16 Universität Ulm TRANSFECTION D'ARNm DE CELLULES PROGÉNITRICES ADULTES POUR UNE RÉGÉNÉRATION TISSULAIRE SPÉCIFIQUE
JP2008535493A (ja) * 2005-04-08 2008-09-04 アルゴス セラピューティクス，インコーポレイティド 樹状細胞組成物および方法
US8263560B2 (en) 2005-04-01 2012-09-11 University Of Maryland Baltimore HPV 16 peptide vaccine for head and neck cancer
US8513008B2 (en) 2004-10-07 2013-08-20 Argos Therapeutics, Inc. Mature dendritic cell compositions and methods for culturing same
US8822182B2 (en) 2001-05-18 2014-09-02 Mayo Foundation For Medical Education And Research Chimeric antigen-specific T cell-activating polypeptides
US8822223B2 (en) 2004-10-07 2014-09-02 Argos Therapeutics, Inc. Mature dendritic cell compositions and methods for culturing same
US10478479B2 (en) 2014-08-01 2019-11-19 Jw Creagene Inc. Method for preparing dendritic cell, dendritic cell prepared thereby, and use thereof
CN112899308A (zh) * 2021-02-05 2021-06-04 北京鼎成肽源生物技术有限公司 一种电转染永生化树突状细胞的方法
US11419925B2 (en) 2013-03-15 2022-08-23 The Trustees Of The University Of Pennsylvania Cancer vaccines and methods of treatment using the same
WO2024008274A1 (fr) 2022-07-04 2024-01-11 Universiteit Antwerpen Modification de lymphocytes t régulateurs

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004063265B4 (de) * 2004-12-29 2012-02-02 Biontech Ag Verfahren zur Bestimmung der Funktion von Nukleinsäuresequenzen und den dadurch kodierten Expressionsprodukten
ES2698600T3 (es) * 2005-12-13 2019-02-05 Univ Pennsylvania Métodos para transfectar ácidos nucleicos en células vivas
US10647960B2 (en) 2005-12-13 2020-05-12 The Trustees Of The University Of Pennsylvania Transcriptome transfer produces cellular phenotype conversion
US9157066B2 (en) 2005-12-13 2015-10-13 The Trustees Of The University Of Pennsylvania Transcriptome transfer produces cellular phenotype conversion
JP2013522309A (ja) * 2010-03-15 2013-06-13 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア 活性化した成熟樹状細胞を調製し保存するシステムおよび方法
US10316332B2 (en) 2011-07-29 2019-06-11 Riken Cell for immunotherapy, including modified nucleic acid construct encoding Wilms tumor gene product
EP2738253A4 (fr) * 2011-07-29 2015-04-22 Riken Cellule utilisable en immunothérapie qui contient un produit de recombinaison de type acide nucléique modifié codant pour un produit du gène de la tumeur de wilms ou pour un fragment de celui-ci, procédé de production de ladite cellule et dudit produit de recombinaison de type acide nucléique
RU2649364C2 (ru) 2011-12-16 2018-04-02 Модерна Терапьютикс, Инк. Составы на основе модифицированного нуклеозида, нуклеотида и нуклеиновой кислоты
US9878056B2 (en) 2012-04-02 2018-01-30 Modernatx, Inc. Modified polynucleotides for the production of cosmetic proteins and peptides
EP3520821A1 (fr) 2012-04-02 2019-08-07 Moderna Therapeutics, Inc. Polynucléotides modifiés pour la production d'agents biologiques et de protéines associées à une maladie humaine
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US10258698B2 (en) 2013-03-14 2019-04-16 Modernatx, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
CN104761644A (zh) * 2014-01-03 2015-07-08 百奇生物科技（苏州）有限公司 大肠杆菌表达的融合蛋白mbp-mart-1及其制备和应用
CN104830794A (zh) * 2015-05-05 2015-08-12 杨光华 基于hpve7抗原的dc细胞、靶向性免疫细胞群及其制备方法和用途
EP4100066A4 (fr) * 2020-02-07 2024-05-01 Rnaimmune, Inc. Composition et procédé de vaccins à arnm contre une infection au nouveau coronavirus
CN118119930A (zh) 2021-08-11 2024-05-31 达冕生物有限公司 抗新型冠状病毒感染的mRNA疫苗的组合物和方法
WO2024097259A1 (fr) 2022-11-01 2024-05-10 RNAimmune, Inc. Composition et procédés pour vaccins à arnm contre de nouvelles infections à coronavirus omicron

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5554528A (en) 1987-08-21 1996-09-10 Board Of Revents Of University Of Colorado Compositions and methods for inhibition of HIV production
US5766902A (en) 1993-08-20 1998-06-16 Therexsys Limited Transfection process

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5124259A (en) * 1989-08-23 1992-06-23 Mitsui Toatsu Chemicals, Inc. Method for electroporation
US6368855B1 (en) * 1996-06-11 2002-04-09 Antigen Express, Inc. MHC class II antigen presenting cells containing oligonucleotides which inhibit Ii protein expression
WO2000052054A2 (fr) * 1999-03-01 2000-09-08 Genentech, Inc. Anticorps destines au traitement et au diagnostic du cancer
NZ516381A (en) * 1999-06-30 2004-03-26 Corixa Corp Lung tumor proteins in the therapy and diagnosis of lung cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5554528A (en) 1987-08-21 1996-09-10 Board Of Revents Of University Of Colorado Compositions and methods for inhibition of HIV production
US5766902A (en) 1993-08-20 1998-06-16 Therexsys Limited Transfection process

Non-Patent Citations (50)

* Cited by examiner, † Cited by third party
Title
ARTHUR, J.F. ET AL., CANCER GENE THER., vol. 4, 1997, pages 17 - 25
AVIGAN, D., BLOOD REV., vol. 13, 1999, pages 51 - 64
BANCHERAU, J.; STEINMANN, R. N., NATURE, vol. 392, 1998, pages 245 - 252
BANCHEREAU, J.; STEINMAN, R.M., NATURE, vol. 392, 1998, pages 245 - 252
BAUM, C. ET AL., BIOTECHNIQUES, vol. 17, 1994, pages 1058 - 1062
BROSSART, P. ET AL., J. IMMUNOL., vol. 158, 1997, pages 3270 - 3276
BUHRING, H. J. ET AL., LEUKEMIA, vol. 5, 1991, pages 854 - 860
CELLA, M. ET AL., CURR. OPIN. IMMUNOL., vol. 9, 1997, pages 10 - 16
DIETZ, A. B.; VUK, P.S., BLOOD, vol. 91, 1998, pages 392 - 398
DIETZ, A.B.; VUK, P.S., BLOOD, vol. 91, 1998, pages 392 - 398
FEUERSTEIN, B. ET AL., J. IMMUNOL. METHODS, vol. 245, 2000, pages 15 - 29
GORDILLO, G. M., TRANSPL. IMMUNOL., vol. 7, 1999, pages 83 - 94
HERBST, B. ET AL., BLOOD, vol. 88, 1996, pages 2541 - 2548
JENNE, L. ET AL., GENE THER., vol. 7, 2000, pages 1575 - 1583
JONULEIT, H. ET AL., GENE THER., vol. 7, 2000, pages 249 - 254
KARIKO, K. ET AL., BIOCHIM. BIOPHYS. ACTA, vol. 1369, 1998, pages 320 - 334
KIM, C. J. ET AL., J. IMMUNOTHER., vol. 20, 1997, pages 276 - 286
KLINMAN, D. M. ET AL., PROC. NATL. ACAD. SCI., vol. 93, 1996, pages 2879 - 2883
KLINMAN, D. M. ET AL., VACCINE, vol. 17, 1999, pages 19 - 25
LARDON, F. ET AL., IMMUNOLOGY, vol. 91, 1997, pages 553 - 559
LU, D. ET AL., CANCER GENE THER., vol. 1, 1994, pages 245 - 252
LU, D. ET AL., CANCER GENE, vol. 1, 1994, pages 245 - 252
LUO, D.; SALTZMAN, W.M., NAT. BIOTECHNOL., vol. 18, 2000, pages 33 - 37
LURQUIN, P.F., MOL. BIOTECHNOL., vol. 7, 1997, pages 5 - 35
MATTHEWS, K.E. ET AL.: "Mol. Biotechnol.", vol. 48
MORSE, M. A. ET AL., CANCER RES., vol. 58, 1998, pages 2965 - 2968
NAIR ET AL., NAT. BIOTECHNOL., vol. 16, 1998, pages 364 - 369
NAIR, S. K. ET AL., NAT. BIOTECHNOL., vol. 16, 1998, pages 364 - 369
PONSAERTS ET AL., JOURNAL OF IMMUNOLOGY, 2002
PORGADOR, A. ET AL., J. EXP. MED., vol. 188, 1998, pages 1075 - 1082
REEVES, M. E. ET AL., CANCER RES., vol. 56, 1996, pages 5672 - 5677
ROMANI, N. ET AL., J. EXP. MED., vol. 180, 1996, pages 83 - 93
ROMANI, N. ET AL., J. IMMUNOL. METHODS, vol. 196, 1996, pages 137 - 151
RONCAROLO, M.G ET AL., J. EXP. MED., vol. 15;193, no. 2, 2000, pages F5 - 9
RONCAROLO, M.G. ET AL., EXP. MED., vol. 15;193, no. 2, 2001, pages F5 - 9
SALLUSTO, F.; LANZAVECCHIA, A., J. EXP. MED., vol. 179, 1994, pages 1109 - 1118
SAWAI, K. ET AL., MOL. GENET. METAB., vol. 64, 1998, pages 44 - 51
SPECHT, J.M. ET AL., J. EXP. MED., vol. 186, 1997, pages 1213 - 1221
SPENCER, S.C., BIOCHEM. AND BIOTECHNOL., vol. 42, 1993, pages 75 - 82
STEPEHNSON, J., JAMA, vol. 281, no. 2, 1999, pages 119 - 122
STROBEL, I. ET AL., GENE THERAPY, vol. 7, 2000, pages 2028 - 2035
TARTE, K.; KLEIN, B., LEUKEMIA, vol. 13, 1999, pages 653 - 663
TENDELOO, V.F.I. ET AL., GENE THER., vol. 5, 1998, pages 700 - 707
VAN BOCKSTAELE, D.; BERNEMAN, Z.N., CYTOMETRY, vol. 41, 2000, pages 31 - 35
VAN TENDELOO V.F.I ET AL., GENE THER., vol. 5, 1998, pages 700 - 707
VAN TENDELOO, V.F.I. ET AL., GENE THER., vol. 5, 1998, pages 700 - 707
VAN TENDELOO, V.F.I. ET AL., GENE THER., vol. 7, 2000, pages 1431 - 1437
VAN TENDERLOO, V. P. ET AL., BLOOD, vol. 98, 2001, pages 49
YING, H. ET AL., NAT. MED., vol. 5, 1999, pages 823 - 827
ZHANG, W. ET AL., HUM. GENE THER., vol. 10, 1999, pages 1151 - 1161

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8822182B2 (en) 2001-05-18 2014-09-02 Mayo Foundation For Medical Education And Research Chimeric antigen-specific T cell-activating polypeptides
US9523077B2 (en) 2004-10-07 2016-12-20 Argos Therapeutics, Inc. Mature dendritic cell compositions and methods for culturing same
US11248209B2 (en) 2004-10-07 2022-02-15 Coimmune, Inc. Mature dendritic cell compositions and methods for culturing same
US10184108B2 (en) 2004-10-07 2019-01-22 Argos Therapeutics, Inc. Mature dendritic cell compositions and methods for culturing same
US9556455B2 (en) 2004-10-07 2017-01-31 Argos Therapeutics, Inc. Mature dendritic cell compositions and methods for culturing same
US8513008B2 (en) 2004-10-07 2013-08-20 Argos Therapeutics, Inc. Mature dendritic cell compositions and methods for culturing same
US8822223B2 (en) 2004-10-07 2014-09-02 Argos Therapeutics, Inc. Mature dendritic cell compositions and methods for culturing same
US8263560B2 (en) 2005-04-01 2012-09-11 University Of Maryland Baltimore HPV 16 peptide vaccine for head and neck cancer
USRE46152E1 (en) 2005-04-08 2016-09-20 Argos Therapeutics, Inc. Dendritic cell compositions and methods
US8501470B2 (en) 2005-04-08 2013-08-06 Argos Therapeutics, Inc. Dendritic cell compositions and methods
US8153425B2 (en) 2005-04-08 2012-04-10 Argos Therapeutics, Inc. Dendritic cell compositions and methods
JP2008535493A (ja) * 2005-04-08 2008-09-04 アルゴス セラピューティクス，インコーポレイティド 樹状細胞組成物および方法
WO2007090647A1 (fr) * 2006-02-08 2007-08-16 Universität Ulm TRANSFECTION D'ARNm DE CELLULES PROGÉNITRICES ADULTES POUR UNE RÉGÉNÉRATION TISSULAIRE SPÉCIFIQUE
US11419925B2 (en) 2013-03-15 2022-08-23 The Trustees Of The University Of Pennsylvania Cancer vaccines and methods of treatment using the same
US10478479B2 (en) 2014-08-01 2019-11-19 Jw Creagene Inc. Method for preparing dendritic cell, dendritic cell prepared thereby, and use thereof
CN112899308A (zh) * 2021-02-05 2021-06-04 北京鼎成肽源生物技术有限公司 一种电转染永生化树突状细胞的方法
WO2024008274A1 (fr) 2022-07-04 2024-01-11 Universiteit Antwerpen Modification de lymphocytes t régulateurs

Also Published As

Publication number Publication date
KR20040025690A (ko) 2004-03-24
CN1701121B (zh) 2013-12-04
EP2308987A2 (fr) 2011-04-13
EP2308987A3 (fr) 2011-04-27
JP2004536598A (ja) 2004-12-09
EP2308987B1 (fr) 2014-09-10
AU2008202507B2 (en) 2011-02-17
WO2003000907A3 (fr) 2003-09-25
CA2451389A1 (fr) 2003-01-03
AU2011201652C1 (en) 2013-03-14
EP1397500B1 (fr) 2014-09-17
AU2011201652B8 (en) 2012-09-20
CA2451389C (fr) 2012-12-11
AU2008202507A1 (en) 2008-06-26
AU2011201652B2 (en) 2012-08-16
BR0210936A (pt) 2004-06-08
AU2011201652A1 (en) 2011-04-28
CN1701121A (zh) 2005-11-23
EP1270732A1 (fr) 2003-01-02
EP1397500A2 (fr) 2004-03-17
JP2011050395A (ja) 2011-03-17

Similar Documents

Publication Publication Date Title
US7547551B2 (en) Transfection of eukaryontic cells with linear polynucleotides by electroporation
AU2008202507B2 (en) Improved transfection of eukaryotic cells with linear polynucleotides by electroporation
Van Tendeloo et al. Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells
Weissman et al. HIV gag mRNA transfection of dendritic cells (DC) delivers encoded antigen to MHC class I and II molecules, causes DC maturation, and induces a potent human in vitro primary immune response
Chiriva‐Internati et al. Efficient generation of cytotoxic T lymphocytes against cervical cancer cells by adeno‐associated virus/human papillomavirus type 16 E7 antigen gene transduction into dendritic cells
Dullaers et al. Induction of effective therapeutic antitumor immunity by direct in vivo administration of lentiviral vectors
US20180078627A1 (en) Method for antigen loading of dendritic cells and vaccine
Ahmed et al. Electroporation as a method of choice to generate genetically modified dendritic cell cancer vaccines
CN1353575A (zh) 用树状细胞一肿瘤细胞或树状细胞一病毒细胞衍生的免疫原在体外诱导抗原特异性t－销细胞
EP3838915A1 (fr) Composition d'immunothérapie antitumorale basée sur des cellules présentatrices d'antigène activées par listeria monocytogenes atténuée, son procédé de préparation et son utilisation
Kikuchi Genetically modified dendritic cells for therapeutic immunity
Philip et al. Dendritic cells loaded with MART-1 peptide or infected with adenoviral construct are functionally equivalent in the induction of tumor-specific cytotoxic T lymphocyte responses in patients with melanoma
Batchu et al. Efficient lysis of epithelial ovarian cancer cells by MAGE-A3-induced cytotoxic T lymphocytes using rAAV-6 capsid mutant vector
JP7519417B2 (ja) Ｓｔｉｎｇ経路を活性化する遺伝子アジュバントの発現のためのウイルスベクター構築物
Met et al. Studies on mRNA electroporation of immature and mature dendritic cells: effects on their immunogenic potential
AU2002319256A1 (en) Improved transfection of eukaryotic cells with linear polynucleotides by electroporation
Artusio et al. Transfection of human monocyte-derived dendritic cells with native tumor DNA induces antigen-specific T-cell responses in vitro.
JP2023011701A (ja) Ｃｄ４０およびｓｔｉｎｇ経路を活性化する遺伝子アジュバントの発現のためのウイルスベクター構築物
Berlyn et al. Developing dendritic cell polynucleotide vaccination for prostate cancer immunotherapy
Markowicz et al. Nonviral transfection of human umbilical cord blood dendritic cells is feasible, but the yield of dendritic cells with transgene expression limits the application of this method in cancer immunotherapy
Ponsaerts Dendritic cell vaccines for immunity and tolerance: mRNA electroporation as a tool for efficient gene transfer
WO2008119024A1 (fr) Cellules modifiées présentant des antigènes et procédés d'utilisation
Van Tendeloo et al. Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: tumor antigen loading of dendritic cells1
Murugaiyan et al. Dendritic cell-based immunotherapy: a promising approach for treatment of cancer
O-SULLIVAN et al. Therapeutic properties of DNA-based fibroblast and dendritic cell vaccines in mice with squamous carcinoma

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2003507290

Country of ref document: JP

Ref document number: 2451389

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2002748810

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 1020037016741

Country of ref document: KR

WWE Wipo information: entry into national phase

Ref document number: 2002319256

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 20028163621

Country of ref document: CN

WWP Wipo information: published in national office

Ref document number: 2002748810

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642